The single substitution I259T, conserved in the plasminogen activator Pla of pandemic Yersinia pestis branches, enhances fibrinolytic activity

Abstract

The outer membrane plasminogen activator Pla of Yersinia pestis is a central virulence factor in plague. The primary structure of the Pla beta-barrel is conserved in Y. pestis biovars Antiqua, Medievalis, and Orientalis, which are associated with pandemics of plague. The Pla molecule of the ancestral Y. pestis lineages Microtus and Angola carries the single amino acid change T259I located in surface loop 5 of the beta-barrel. Recombinant Y. pestis KIM D34 or Escherichia coli XL1 expressing Pla T259I was impaired in fibrinolysis and in plasminogen activation. Lack of detectable generation of the catalytic light chain of plasmin and inactivation of plasmin enzymatic activity by the Pla T259I construct indicated that Microtus Pla cleaved the plasminogen molecule more unspecifically than did common Pla. The isoform pattern of the Pla T259I molecule was different from that of the common Pla molecule. Microtus Pla was more efficient than wild-type Pla in alpha(2)-antiplasmin inactivation. Pla of Y. pestis and PgtE of Salmonella enterica have evolved from the same omptin ancestor, and their comparison showed that PgtE was poor in plasminogen activation but exhibited efficient antiprotease inactivation. The substitution (259)IIDKT/TIDKN in PgtE, constructed to mimic the L5 region in Pla, altered proteolysis in favor of plasmin formation, whereas the reverse substitution (259)TIDKN/IIDKT in Pla altered proteolysis in favor of alpha(2)-antiplasmin inactivation. The results suggest that Microtus Pla represents an ancestral form of Pla that has evolved into a more efficient plasminogen activator in the pandemic Y. pestis lineages.

FIG. 1.

Model of Pla structure (23) and location of residue Thr259. Side (top drawing) and top (bottom drawing) views of the transmembrane β-barrel are shown. L1 to L5 are the surface loops. Catalytic residues Asp84, Asp86, Asp206, and His208 are indicated in green, Thr259 is in red, and the autoprocessing site Lys262 is in yellow. OM is the outer membrane. (C) Amino acid sequence of residues 254 to 273 at L5 and the termini of β-strands 9 and 10 in Pla, Microtus Pla, and PgtE are shown.

FIG. 2.

Expression and isoform patterns of Pla and the Pla derivatives in recombinant bacteria. (A) Pla and Pla T259I were expressed in Y. pestis KIMD34, and the Western blot assay shows the reactivity of whole-cell samples with anti-Pla antiserum. (B) Expression and isoforms of the Pla proteins in cell envelope preparations from recombinant E. coli. The protein constructs are indicated above the lanes, and the migration distances of the α, β, and γ isoforms of the mature Pla molecule and of nonprocessed pre-Pla are indicated on the left. The plus sign denotes samples boiled for 10 min before electrophoresis, and the minus sign denotes unboiled samples.

FIG. 3.

Fibrinolysis by recombinant Y. pestis KIM D34 and E. coli XL1. Bacteria (10⁷ cells) in buffer were pipetted onto fibrin plates containing 5 μg/ml Plg, and dissolution of the clot was assessed after incubation for 20 h (E. coli) or 48 h (Y. pestis) at 37°C. (A) Bacterial numbering: 1, Y. pestis KIM D34(pSE380); 2, Y. pestis KIM D34(pMRK1); 3, Y. pestis KIM D34(pPlaT259I). (B) Bacterial numbering: 1, E. coli XL1(pSE380); 2, E. coli XL1(pMRK1); 3, E. coli XL1(pPlaT259I); 4, E. coli XL1(pMRK1.51); 5, E. coli XL1(pMRK3); 6, E. coli XL1(pMRK3.51).

FIG. 4.

Plasminogen activation by recombinant Y. pestis KIM D34 and E. coli XL1 and plasmin inactivation by recombinant E. coli XL1. (A and B) Cumulative, initial plasmin formation by bacteria tested at two Plg concentrations (20 and 2 μg/ml). The expression host, protein constructs, and Plg concentrations are indicated. (C and D) Plasmin formation by bacteria after incubation for up to 22 h with Plg. Plasmin formation was analyzed by adding a chromogenic plasmin substrate at the indicated time points and measuring absorbance after 15 min. (E) Inhibition of plasmin activity by recombinant E. coli. Plasmin was incubated for 30 min with the bacteria, after which the plasmin substrate was added and the absorbance was measured after 90 min. The assays were repeated at least three times, and results of a representative assay with duplicate samples are shown.

FIG. 5.

Degradation of plasminogen by Pla and PgtE variants expressed in recombinant E. coli XL1. Plg degradation by the bacteria was analyzed by Western blotting with anti-Plg antibody (A) and anti-Plg catalytic domain antibody (B). The migration distances of Plg, plasmin heavy chain (PlnH, apparent size of 70 kDa), plasmin light chain (PlnL, 25 kDa), and angiostatins (Ang) are shown on the left. PlgX indicates a fragment of Plg produced by the bacteria. The Pla and PgtE constructs expressed in E. coli XL1 and uPA-treated Plg are indicated above the lanes, incubation times in panel A are indicated below the lanes. In panel B, the incubation time was 2 h.

FIG. 6.

Degradation and inactivation of α₂AP by recombinant E. coli XL1. (A) Degradation of α₂AP after a 2-h incubation with bacteria, as analyzed by Western blotting. The protein constructs expressed in E. coli XL1 are indicated above the lanes. The migration distance of α₂AP is shown on the left, and the arrow indicates a cleavage product of α₂AP. (B) Inactivation of α₂AP by bacteria. The Pla and PgtE constructs expressed in E. coli XL1 are indicated. Bacteria were incubated with α₂AP for 2 h, after which plasmin and its chromogenic substrate were added and plasmin activity was measured after 15 min. The assay was repeated three times, and results of a representative assay with duplicate samples are shown.